American-Eurasian Journal of Scientific Research 15 (4): 128-139, 2020ISSN 1818-6785© IDOSI Publications, 2020DOI: 10.5829/idosi.aejsr.2020.128.139

Corresponding Author: Warkaw Merachew, School of Veterinary Medicine, College of Agriculture and Veterinary Medicine,Jimma University, Jimma, Ethiopia.

128

The Mechanisms of Action, Effects and Resistance Profiles ofAnthelmentics Used in the Treatment of Ovine Fasciolosis

Warkaw Merachew

School of Veterinary Medicine, College of Agriculture and Veterinary Medicine,Jimma University, Jimma, Ethiopia

Abstract: Fasciolosis is the common parasitic disease affecting both humans and animals all over the world.Fasciola hepatica and Fasciola gigantica are the two liver flukes commonly reported to cause fasciollosis insheep. The disease affects the liver parenchyma and bile ducts of numerous animals. It is one of the majorcauses of liver condemnations at abattoirs and interferes with fertility and fecundity. The transmission of thedisease is enhanced by cullucoide snail hosts. To prevent this serous disease, anthelmintics are recommended.Among these anthelmintic drugs, Albendazole and Triclabendazole are in line of curing the disease.Especially, the latter is the choice of drug against both species of Fasciola infection in humans and animals.However, reports showed that the disease is now developing resistance in some regions of the world.Hence, this review was focused on the mechanisms, effects and resistance profiles of anthelmintic drugs thatare used against fasciollosis.

Key words: Fasciola Resistance Anthelmintic Mechanism of Action Prevention

INTRODUCTION condemnations at abattoirs and interferes with fertility

Ethiopia owns about 23.6 million sheep and 23.3 that produces considerable loss in sheep and cattlemillion goats [1] that are mainly kept by smallholder worldwide [7]. In Ethiopia, ovine fascioliasis lossesresource poor farmers. In Ethiopia, small ruminants are annually an estimated ETB of 48.4 million due to mortality,important sources of income for rural communities whose productivity (weight loss and reproductive wastage)livelihood is largely based on livestock production [2, 3]. and liver condemnation at slaughter [8]. However, sheep production in the country is hindered by Control and prevention measures of fascioliasisvarious factors including animal health constraints, should be done on a preventative rather than curative.inadequate nutrition, poor husbandry systems and lack of Three effective control strategies have been used whicheffective veterinary services [4]. are: use of anthelmintic to reduce the number of liver fluke

Fascioliasis is among the important parasitic diseases in the definitive hosts and the number of fluke eggs onin tropical and subtropical countries which limit the pasture, reduce the number of intermediate host andproductivity of ruminants. Fasciola hepatica and reduce exposure to infection by managing fluke proneFasciola gigantica are the two liver flukes commonly areas. Anthelmintics are drugs that are used to treatreported to cause fascioliasis in sheep [5]. Fasciola infections with parasitic worms. This includes both flatspecies infects mammals worldwide, mainly ruminants, worms, e.g., flukes and tapeworms and round worms, i.e.,but also humans can become infected. In ruminants nematodes. They are of huge importance for humanand especially in sheep, the infection reduces feed tropical medicine and for veterinary medicine. Broadconversion, growth and meat and milk production. spectrum anthelmintics are effective against parasitic flatFascioliasis is a disease that affects the liver parenchyma worms and nematodes [9]. and bile ducts of numerous animals, including humans, The correct time to use anthelmintics is based onwhich causes economic losses and threatens public weather and climate conditions. Drugs play a crucial rolehealth. Moreover, it is one of the major causes of liver in the control of fascioliasis. More frequent treatments are

and fecundity [6]. Fascioliasis is a very serious disease

Am-Euras. J. Sci. Res., 15 (4): 128-139, 2020

129

necessary if you use drugs that are only effective against massive, but relatively short-term, intake of metacercariae.advanced mature flukes aged 12-16 weeks or older. Death usually results from blood loss due to hemorrhageUsing TCBZ based flukicides, the most effective drug and tissue destruction caused by the migratory juvenileagainst both early mature and adult liver flukes. The best flukes in the liver resulting in traumatic hepatitis.control measures may be achieved if this drug use three Diagnosis of Fasciolosis is based on clinical sign, grazingtimes yearly. Namely; August/September: to prevent history and seasonal occurrence, examination of feces bypasture from contamination and to eliminate adult flukes laboratory tests and post mortem examination [15].came from autumn and winter. January/February: tocompletely remove of flukes picked up during late spring Treatment of Ovine Fasciolosis: Treatment of infectedand early summer. April/May: to remove flukes picked up animals will largely depend on the correct use ofduring summer and early autumn [10]. Several drugs have appropriate and registered anthelmintic. Several drugsbeen proposed for the treatment of fascioliasis mainly have been proposed for the treatment of fascioliasisbithionol, praziquantel and benzimidazole (BZD) family of mainly, bithionol, praziquantel and benzimidazole (BZD)anthelmintics such as Triclabendazole (TCBZ) and family of anthelmintics such as triclabendazole (TCBZ)Albendazole (ABZ) [11]. and albendazole (ABZ). Triclabendazole is the most

In Ethiopia, Fascioliasis infections in sheep are effective anthelmintic drug which can be destroys or killscontrolled by application of Albendazole and all stage of Fasciola spp. Fasciolosis may be controlled byTriclabendazole. However, drug efficacy can be reducing the populations of the intermediate snail host ornegatively affected by various factors such as under by appropriate anthelminthic treatment and the populationdosing, resistance arising from the exclusive use of drugs of snail should be destroyed by applying Molluscicideof the same mode of action for long periods of time, the and destroying the environment that suit for snail’suse of substandard quality drugs and inappropriate use reproduction [15].of the drugs. Misuse and smuggling of anthelmintics inmany forms, such as illegal trading in open markets and Anthelmintics: Anthelmintics are drugs that are used toirrational administration, are widespread in the study area treat infections with parasitic worms. This includes bothand most parts of the country. This is due to an absence flat worms, e.g., flukes and tapeworms and roundof strong regulatory system on the use of anthelmintics worms, i.e., nematodes. They are of huge importance foruse. In addition, methods that can preserve and prolong human tropical medicine and for veterinary medicine.the efficacy of anthelmintics and prevent the emergence Broad spectrum anthelmintics are effective againstof anthelmintics resistance, are very low all over the parasitic flat worms and nematodes [9]. Anthelminticscountry [12, 13]. Hence, this review was focused on the expel parasites from the body by either stunning ormechanisms, effects and resistance profiles of killing them and without causing significant damageanthelmintic drugs that are used against fasciollosis. to the host. Many modern anthelmintics are effective

Overview on Ovine Fascioliasis: Fascioliasis is a number are efficacious against arrested or dormant larvaeworldwide zoonosis caused by Fasciola spp. and is often [17].neglected despite its common occurrence in endemic Three classes of anthelmintics are available for use inareas and it is caused by two species of parasitic ruminants. These are Macrocyclic lactones (MLs),flatworms or trematodes that mainly affect the liver. Benzimidazoles (BZs) and the ImidazolthiazolesFasciolosis is one of the major constraint factors for Tetrahydropyrimidines (I-T). These three substanceovine production development in Ethiopia by inflecting classes of anthelmintics vary in their mechanisms ofdirect and indirect loss at different parts of the country. action. An ideal anthelmintic should be efficient againstOvine fasciolosis is an economically important parasitic all parasitic stages of a particular species, non-toxic to thedisease of sheep caused by trematodes species of the host, rapidly cleared and excreted by the host, easilygenus Fasciola, which migrate in the hepatic parenchyma administered and the cost of an anthelmintic should beand establish and develops in the bile ducts [14]. reasonable. Anthelmintics are generally used in two ways,

In Ethiopia, both species co-exist at different namely, therapeutically, to treat existing infections oraltitudes. The snails of the genus lymnae are mainly clinical outbreaks, or prophylactically, in which the timinginvolved as an intermediate host in the life cycle of of treatment is based on knowledge of the epidemiologyfasciolosis. This Fasciola disease has three phases of [18]. The effect of anthelmintics treatment is manifested inclinical sign acute, sub-acute and chronic forms. Acute many ways, such as enhanced growth rate, reproductivefasciolosis occurs as disease outbreak following a performance and wool production [19].

against both adults and larval stages and an increasing

Am-Euras. J. Sci. Res., 15 (4): 128-139, 2020

130

Fig. 1: Morphology of Fasciola species [14]

Fig. 2: The life cycle of Fasciola species [14]

Table 1: List of Currently Available Drugs for the treatment of Fascioliasis in Sheep worldwideFlukicides: Active Compound(s) Method of Administration Available Age of F. hepatica Killed Reports of Resistance On-FarmTCBZ and TCBZ-based combinations Oral, pour-on From early immature 30 casesAlbendazole Oral, intraruminal From adult 3 casesClorsulon Injectable, oral From adult; from late immature for oral 3 casesClosantel Pour-on, injectable, oral From late immature 1 caseNitrox nil Injectable From adult 1 caseSource: [16]

Benzimidazole:Benzimidazoles (BZD) are broad-spectrum their diverse biological activity and clinical applications,anthelmintic compounds widely used in human and they are remarkably effective compounds both withveterinary medicine to control nematode, cestode and respect to their inhibitory activity and their favorabletrematode infections. They are a group of anthelmintic selectivity ratio. The BZs compounds currently marketeddrugs commonly used in ruminants for the treatment and as anthelmintics can be grouped as BZD thiazolyls,prevention of infections caused by helminthes. BZD methylcarbamates, pro-BZD and halogenated BZDBenzimidazole is the heterocyclic compound formed from thiols. Only a few molecules within the BZD chemicalbenzene and imidazole ring containing nitrogen, oxygen, family demonstrated activity against the trematode,sulphur and its derivatives are of wide interest because of Fasciola hepatica [20, 21].

Am-Euras. J. Sci. Res., 15 (4): 128-139, 2020

131

Fig. 3: Molecular structure of triclabendazole [28]

Fig. 4: Chemical structure of TCBZ and its metabolites [33]

BZD are effective against a broad range of parasites fasciolosis. TCBZ is usually the anthelmintic of choiceand also have wide safety margins, working at dosages of against Fasciola infection in sheep, as this drug has highmg/kg bodyweight [22]. Their mode of action appears to activity against both adult and down to 1 week oldbe mediated through binding to -tubulin within the juvenile flukes [29]. TCBZ has shown no activity againstparasite, thus inhibiting the formation of microtubules that nematodes. It differs from the other benzimidazoleare central to the form and function of the parasite’s cells. anthelmintics (e.g., albendazole, mebendazole) currentlyThis prevents various essential cellular processes such as used in humans since these compounds have selectivethe transport of secretory granules and enzymes in the activity against nematodes and have no significantcell cytoplasm, resulting in cell lysis, with knock-on activity against flukes and other trematode helminthesdetrimental effects on motility and feeding [23]. The BZs [30].bind to helminth â-tubulin and prevent the polymerizationof the microtubules and exert its effects by interfering with Pharmacokinetics and Pharmacodynamics ofcell division and the glucose uptake [24, 25]. Triclabendazole

Triclabendazole: Triclabendazole (6-chloro-5(2-3- administration, triclabendazole is absorbed from thedichlorophenoxy)-2-methyl-thio-benzimidazole), a gastrointestinal tract; absorption is increased twofold tohalogenated benzimidazole-thiol derivative, is one of the threefold when triclabendazole is taken after a fatty meal.major anthelminthic drugs used to control fasciolosis in Triclabendazole and its metabolites attain highsheep and cattle. TCBZ was first introduced as a flukicide concentrations in the biliary tract, through which they areduring the early 1980s. It has an efficacious (> 98%) drug excreted back into the intestine over a period of severalfor both mature and immature flukes and has been used to days; less than 1% of orally administered triclabendazoletreat and control fascioliasis [26]. Due to its efficacy for is distributed into breast milk [31]. Triclabendazole isimmature flukes TCBZ is the best drug of choice among oxidized to sulfoxide (the primary metabolite) and sulfoneother anthelminthic agents and considered as an Achilles (present in lesser amounts) over the first 24 hoursheel in the overall control of liver fluke [27]. following oral administration.

Triclabendazole is flukicidal BZD compounds Briefly, TCBZ is completely removed from the portalextensively used in veterinary medicine. Effective blood by the liver and cannot be detected in the plasma.strategies for the control of fasciolosis are mainly based It is oxidized to the sulphoxide (TCBZ.SO) and sulphoneon the use of drugs. TCBZ (Fasinex®, Novartis) is (TCBZ.SO2) metabolites, which are the main metabolitesworldwide one of the most used drugs for the control of present in the plasma. Hydroxylation of TCBZ and its two

Pharmacokinetics of Triclabendazole: Following oral

Am-Euras. J. Sci. Res., 15 (4): 128-139, 2020

132

metabolites takes place in the liver, too, giving rise to the Pharmacodynamics of Triclabendazole: The extensivecorresponding hydroxy metabolites, which are excreted in metabolism of TCBZ by the host means that (potentially)the bile [32]. the adult fluke is exposed to a number of different forms

The flavinmonooxygenase (FMO) pathway is the of TCBZ. Moreover, each of the compounds is capable ofmain pathway involved in the conversion of TCBZ to entering the fluke via diffusion, entry being closely relatedTCBZ.SO, while it contributes equally with the to their lipophilicity. TCBZ, TCBZ.SO and TCBZ.SO2cytochrome P450 (P450) enzyme system to the demonstrate a similar ability to diffuse into the fluke andsulphonation of TCBZ.SO to TCBZ.SO [34, 35]. It has their level of diffusion is higher than that for the2

been shown that the rumen microflora are capable of corresponding hydroxyl compounds [34]. carrying out the sulpho reduction of TCBZ.SO and Entry of TCBZ compounds into the fluke has beenOH-TCBZ.SO to TCBZ and OH-TCBZ, respectively, shown to take place principally by means of diffusionsuggesting that the rumen can act as a reservoir of TCBZ across the tegument, rather than by oral ingestion, a resultcompounds. This could serve as a slow-release system that is surprising, perhaps, given the strong binding offor the further availability of TCBZ in the digestive tract, the metabolites to plasma proteins. Two approaches havefrom where it could be absorbed and passed to the liver been used to confirm this idea, one pharmacological and[35]. TCBZ can also be oxidized to TCBZ.SO by digestive the other one is morphological. Both made use of flukesmicroflora prior to its absorption or by the intestinal wall that had been ligatured to prevent oral entry of drug.during absorption. It is evident, that the mechanisms of Following incubation in TCBZ.SO, its concentration in theTCBZ metabolism are complex, but serve (together with fluke was similar, irrespective of whether the fluke hadthe strong binding to plasma proteins) to maintain active been ligatured or not. When an excess of bovine serumconcentrations of TCBZ compounds in the host for albumin (BSA) was added to the incubation medium, inconsiderable periods of time and this undoubtedly order to allow most of the drug to bind to it, theenhances drug efficacy. Elimination of the drug is done concentration of TCBZ.SO was reduced (by 85%) in boththrough Fecal-Approximately 95% of orally administered ligatured and non-ligatured flukes [34]. triclabendazole (unchanged or as the primary metabolite) A parallel morphological study has been carried out,is excreted in the feces and renal approximately 2% is to compare drug-induced changes to the tegument andexcreted in the urine [36]. gut following incubation with TCBZ.SO. Disruption to the

Triclabendazole is marketed in combination with other tegument, as assessed by scanning electron microscopyanthelmintics and there may be interactions between the (SEM), was similar in ligatured and non-ligatured flukes,drugs that affect its pharmacokinetics. One such indicating that restricting the oral uptake of drug does notcombination is TCBZ plus ivermectin. Ivermectin itself has affect the ability of TCBZ.SO to enter the fluke and exertno activity against trematodes such as Fasciola [37], but its effect. Incubation with TCBZ.SO in the presence of ana recent study has shown how it can affect the excess of BSA led to a reduction in the level of tegumentaldisposition of TCBZ and its metabolites [38]. disruption. In all experiments, the gut remained unaffected

Fig. 5: Decreased efflux of TCBZ and TCBZ.SO in TCBZ.SO2 has been shown to have some activity in itsTCBZ-R flukes following co-incubation with own right in vivo: it caused a 41% reduction in wormIvermectin [26] burden against a juvenile fluke infection in sheep [29].

by TCBZ.SO action, suggesting that the oral uptake ofdrug plays only a (very) minor role in drug entry [39].

In terms of drug action, the fluke is known to play amore active role than simply being subject to thepassive uptake of drug and its diffusion to the site ofaction, as it has been shown to be capable of metabolizingTCBZ to TCBZ.SO and TCBZ.SO to TCBZ.SO2 [34, 40].The precise mechanism remains to be fully elucidated, butthere is more evidence for an action against microtubulesand microtubule-based processes than for otherpossibilities, such as against energy metabolism orneuromuscular co-ordination. Since TCBZ.SO is the mainmetabolite present in both blood plasma and bile, it hasbeen assumed to be the active form of TCBZ. Moreover,

Am-Euras. J. Sci. Res., 15 (4): 128-139, 2020

133

The level of surface disruption induced by the and/or inhibit the association of GTP-Ras withthree compounds varied from region to region and adenylatecyclase [44]. Most of the studies on theoverall was similar, but that caused by TCBZ was slightly mechanism of action of TCBZ have been carried out withgreater than that of produced by the two metabolites. TCBZ.SO. The precise mechanism remains to be fullyInternal changes observed were greatest following elucidated, but there is more evidence for an actiontreatment with TCBZ.SO2 and, while TCBZ.SO was also against microtubules and microtubule-based processesdisruptive, TCBZ was far less disruptive. Combining the than for other possibilities, such as against energyresults for surface and internal changes, the order of metabolism or neuromuscular co-ordination, [29].severity of disruption was TCBZ.SO2, .TCBZ.SO andTCBZ. So, TCBZ.SO2 may well contribute to drug action Efficacy of Fascilocide Drugs: Not all compounds arein vivo and is not the inactive metabolite that it was equally effective against all stages of development ofpreviously thought to be. It may further disrupt flukes Fasciola species in the body. Early effective treatment isalready affected by TCBZ.SO. So drug action may be the the cornerstone of helminthes control. Effective drugs willcombined effect of several metabolites, rather than being exhibit a selective toxicity for the pathogen as compareddue to a single compound [41]. to the host [45]. For the treatment of acute Fasciolosis, it

Mechanism of Action of Triclabendazole: TCBZ is a BZD the juveniles that damage the liver parenchyma. Forderivative and all available evidence from gastrointestinal chronic disease, a compound active against adult fluke isround-worms indicates that BZD anthelmintics bind to required. Triclabendazole (Fasinex) is considered as the

- and -tubulins within the cells of the parasite, causing most common drug due to its high efficacy against adultdisruption of vital processes, such as feeding and as well juvenile flukes. It is ovicidal and well kills anydigestion. Several morphological studies of the effects Fasciola species eggs present in the bile duct or theof TCBZ and its active metabolites on Fasciola species, alimentary tract at the time of treatment [7]. The efficacy ofhave examined on the tegument, vitellaria and testis of the main compounds used to treat fasciolosis and theirthe fluke; all three tissues showed significant signs of spectrum of activity against flukes of different ages isUltra structural disruption, consistent with inhibition of summarized in Figure 6. microtubule-based processes which, in turn, would Monitoring the therapeutic efficacy of anthelminticsprevent the movement of secretory bodies from the cell is therefore a fundamental component of treatmentbodies to the tegmental surface [26]. There is inhibition of strategies. As the parasite evolves continuously tomitosis in the vitelline and spermatogenic cells; disruption develop resistance to drugs, continuous globalof transport processes in the tegument (the outer layer of monitoring and reporting of drug efficacy and parasitea trematode), which leads to progressively severe damage resistance are needed [4]. Only a program of surveillanceof the tegmental surface [29]. There is also a concurrent of therapeutic efficacy and anthelmintic drug resistanceloss of tubulin immuno sustaining in the tegmental will allow detection of changing patterns of parasitesyncytium, further implicating an interaction with tubulin susceptibility and timely revision of national and globalas the primary mode of action of TCBZ [42]. parasite treatment policies [30].

Fasciolicidal not only against the adult wormspresent in the biliary ducts, but also against the immature Efficacy of Triclabendazole: TCBZ was first introduced aslarval stages of Fasciola migrating through the hepatic a flukicide during the early 1980s. It has an efficacious (>parenchyma; the mechanism of action is not thoroughly 98%) drug for both mature and immature flukes and hasunderstood; however, triclabendazole is shown to been used to treat and control fasciollosis [26]. Due to itspenetrate into liver flukes by transtegumentary absorption efficacy for immature flukes TCBZ is the best drug offollowed by inhibition of the parasite's motility, probably choice among other anthelminthic agents and consideredrelated to the destruction of the microtubular structure, as an Achilles heel in the overall control of liver fluke [27].resulting in the death of the parasite; the immobilizing This over-reliance on TCBZ to treat sheep and, to a lessereffect is paralleled by changes in the parasite's resting extent, cattle, has resulted in selection for flukes resistanttegumental membrane potential, strongly inhibiting the to TCBZ. Studies on the efficacy of TCBZ have centeredrelease of proteolytic enzymes, a process that appears mainly on field trials involving pre- and post-treatmentcritical to the survival of the parasite [43]. Recently, TCBZ fluke counts, faecal egg count reduction (FECRT) andwas reported to inhibit adenylatecyclase activity in yeast clinical chemistry [46].

is essential to choose a product highly effective against

Am-Euras. J. Sci. Res., 15 (4): 128-139, 2020

134

Fig. 6: Flukicides and their efficacy [7]

Unlike other BZD compounds, the halogenated under dosage and inefficiency against arrested or dormantderivative TCBZ has been shown to have an excellent larva may be some of the reasons for the reducedefficacy against the mature and immature stages of efficacy and for the increasing development of AR [17].Fasciola species. However, TCBZ activity appears to be The availability of fake and adulterated anthelmintics inrestricted to the liver fluke and the lung fluke, the drug market in many of the under developed countriesParagonimus species. Since the drug is inactive against is another important contributing factor to thenematodes, cestodes and other trematode parasites. development of resistant strains. Generally, the presenceThe intensive use of TCBZ in endemic areas of of AR depends upon factors associated with the host, thefascioliasis has resulted in the development of liver flukes parasite, type of anthelmintic, animal management andresistant to this compound, which is considered a major climatic characteristics, thus increasing the difficulties forproblem for veterinary therapeutics [46, 47]. the establishment of preventive measures, which should

Anthelmintics Resistance: Anthelmintic resistance (AR)is defined by Køhler as genetically transmitted loss of Triclabendazole Resistance: Resistance tosensitivity of a drug in worm populations that were triclabendazole was first described in the United Kingdompreviously sensitive to the same drug and the heritable (UK) in the late 1990’s and has now been reported onability of the parasite tolerate a normally effective dose of numerous occasions in fluke populations affecting sheepthe anthelmintic” [48]. Since resistance is inherited, the and cattle. Mechanisms involved in the development ofsurviving worms will pass their resistance alleles to their resistance to the TCBZ can result from changes in theprogeny. In a worm population, alleles coding for target molecule, in drug uptake/efflux mechanisms and inresistance will be present as a result of mutations, also in drug metabolism. The failure of TCBZ to kill liver flukeunexposed populations. Resistance will develop if there could be due to several factors ranging from problematicare survival advantages for parasites carrying these drug delivery, reduced host liver metabolism of TCBZ toalleles. Treating worms with drugs corresponding to the active pro-drug, or management practices that select for“resistance” alleles will give these worms an advantage TCBZ resistant parasites [50].and the frequency of resistant worms in the population Triclabendazole resistance (TCBZ-R) has likelywill increase. The frequency of alleles coding for appeared due to poor understanding of liver fluke biologyresistance at the time of exposure to a drug will be by farmers and con-founding factors, such as incorrectimportant for the rate of the development of a resistant dosing, inappropriate product choice and lack of testingpopulation [48]. for efficacy. The high frequency of TCBZ use, effectively

Frequent dosing (extensive use), adoption of TCBZ mono therapy with no anthelmintic rotation, was acommon management and therapeutic strategies, long- major contributing factor towards the development ofterm utilization, inappropriate handling, rapid reinfection, TCBZ-R [47]. Resistance of TCBZ described in different

vary according to the animal production systems [49].

Am-Euras. J. Sci. Res., 15 (4): 128-139, 2020

135

parts of the world mostly in European countries such as regarded as successful if there is a 95% reduction in flukeNetherland, Britain, Russia, Scotland and main land of egg counts by 14 days post-treatment. However, it isEurope [29]. known that eggs can be stored in the gall bladder for

Methods for Detection of Triclabendazole Resistance: the flukes have been successfully removed; this can leadDifferent methods, both in vivo and in vitro methods, to false positive results. Moreover, egg production byhave been used to detect and monitor AR. Faecal egg flukes ceases within 2 days of successful TCBZ treatmentcount reduction test (FECRT) is the most used in vivo [56, 57].method and gives an estimation of the efficacy of the drug Other disadvantages of the test include the fact thatby comparing the egg counts pre and post treatment. there is no standard method (i.e. sedimentation, floatation,The accuracy of the method depends on a correlation individual or composite samples) and faecal egg countsbetween egg counts and worm burdens which is not are not related to fluke numbers; also, for diagnosis ofalways present. From in vitro methods the egg hatch infection, it only detects patent infections and eggassay (EHA) was the most commonly used which shedding is irregular. Fluke counts may be more accurateis first described by Le Jambre for the detection of but are not always carried out and this data runs intoBZD- resistance. Modification of the original method is problems of trial design and how the flukes are recovered.developed by [51] and the method is mostly used for the The FECRT is often used for field cases, though it suffersdetection of possible BZD resistance in sheep and horses from the problems outlined above and is not always linked[52]. to fluke count data [57].

In vitro Method: The EHA is based on the ovicidal Management Strategies to Prevent Development ofproperties of some BZs and on the capacity of eggs fromresistant isolates to embrionate and hatch at higherconcentrations than those ones from a susceptible isolate[53]. Although the EHA was originally designed to detectAR in gastrointestinal nematodes some studies have beencarried out with F. species eggs from gall bladder and/orfaeces using TCBZ and their sulphoxide metabolites [54].

A commercial formulation of TCBZ (Fasinex®) dilutedin dimethyl sulfoxide (DMSO) was used to carry out theEHAs. The concentration of TCBZ in this commercialformulation was 50 mg/ml. Dilutions of 10, 40, 200, 1000and 5000 µg/ml were prepared to obtain a finalconcentration in the wells of 0.05, 0.2, 1, 5 and 25 µg/ mlafter adding 10 µl of each dilution to a total volume of2 ml. In all EHAs, control wells with 10 µl of DMSO wereincluded. Eggs from faeces were obtained bysedimentation, F. species eggs were directly recoveredfrom the gall bladder and washed several times with tapwater [53].

In vivo Method: The main method used to identifyTCBZ-R in the field has been the faecal egg countreduction test (FECRT), with the recommended post-treatment sample collection time point at 21 days [55].Other studies using experimental infections have used 14days for post-treatment sample collection, which may notallow sufficient time for all eggs from dead parasites topass out of the gall bladder and be excreted. The FECRTis probably most often used, with drug treatment being

several weeks, so they may still be present, even though

Triclabendazole ResistanceUse of Other Drugs and Their Combinations: Thechemotherapy options for the control of TCBZ-R flukesare, depending on the host species, treatment withclorsulon, nitroxynil, closantel, Albendazole, oroxyclozanide [58]. The use of dual-active flukicides hasbeen recommended to control Fasciola infection that wasresistant to TCBZ. Dual formulations have a synergisticeffect (i.e., have greater efficacy than the sum of theactives), this may increase the lifespan of the respectiveactives. Synergy has been seen with several dual-activeflukicides (e.g., TCBZ+ clorsulon or TCBZ+luxabendazole) against TCBZ-R fluke in sheep [59].

Vaccination: An alternative approach to control TCBZ-Rwould be the development of a livestock vaccine forFasciola species infection, which would reduce flukeburdens irrespective of the drug resistance. However, nocommercial liver fluke vaccine exists, although severalexperimental vaccines for livestock are underdevelopment. No vaccine has shown reproducibly highenough efficacies (> 60%) in sheep to warrant commercialproduction, although the leucine amino-peptidase (LAP)vaccine has shown high efficacy (up to 89%) [60].Thus, until a new anthelmintic is developed that kills alldevelopmental stages, including the early immature fluke,a vaccine is the only alternative treatment that couldprovide ongoing control of fluke infections in livestock inregions where TCBZ-R is endemic [61].

Am-Euras. J. Sci. Res., 15 (4): 128-139, 2020

136

Fig. 7: Integrated Parasite Management [16]

Integrated Parasite Management for Farms: The Ethno-Veterinary Medicine: Ethno-veterinary medicinemanagement practices on farms generally rely solely upon covers people’s knowledge, skills, methods, practices andanthelmintics and appear to have contributed to the beliefs about the care of their animals. It provides valuabledevelopment of resistance [62]. Management practices alternatives to and complements for veterinary medicine.must change to preserve the longevity of existing It can play an important role in grassroots developmentflukicides, because the likelihood of any new flukicides which seeks to empower people by enhancing the use ofcoming to market in the near future is low [60]. their own knowledge and resources. Many indigenousThroughout the year, there are periods in which the risk veterinary beliefs and practices persist in a wide majorityof fluke infection is higher and these periods fluctuate of stock raisers and farmers, particularly in the developingdepending upon location and prevailing climatic countries.conditions [63]. A number of medicinal plants have been used to treat

Well-executed strategic treatments will minimize the parasitic infections in man and animals. Among the mostneed for further treatments throughout the year and, common medicinal plants which have anthelmintics effecttherefore, help to preserve the efficacy of existing for Fasciola species are, Artemisia species plant is theflukicides. Regular drug efficacy testing, using FECRT to most commonly one used in different parts of the world.preserve the efficacy of existing flukicides is essential to This plant can be a promising future for the control ofallow producers to avoid using products with reduced liver flukes which had previously shown resistance toefficacy and prevent economic losses resulting from synthetic drugs [60].unidentified resistance [60]. Flukicides should alwaysbe administered according to the product specifications CONCLUSIONSand best-practice methods, which include: weighingindividual animals or the heaviest in the herd to determine Treatment of infected animals with Fasciola is largelydose, calibrating drench equipment before use and dependent on the correct use of appropriate andduring treatments, selecting the most potent formulations registered anthelmintic. Several drugs have beenof product and regularly rotating effective products. proposed for the treatment of fascioliasis mainly,In addition, we must also look at pastures, drinking water bithionol, praziquantel and benzimidazole (BZD) family ofand irrigation can be better managed to decrease the anthelmintics such as triclabendazole (TCBZ) andlikelihood of Fasciola infection. Pasture management can albendazole (ABZ). Triclabendazole is the most effectiveallocate low-risk pastures (such as newly sown paddocks, anthelmintic drug which can be destroys or kills all stagehay, or silage paddocks) to young animals during the of Fasciola spp. Fasciolosis may be controlled byhigh-risk periods, to limit the chances of parasite reducing the populations of the intermediate snail host ortransmission [60]. by appropriate anthelminthic treatment and the population

Am-Euras. J. Sci. Res., 15 (4): 128-139, 2020

137

of snail should be destroyed by applying Molluscicide 5. Keyyu, J.D.J., N.C. Monrad, Kyvsgaard andand destroying the environment that suit for snail’s A.A. Kassuku, 2005. Epidemiology ofreproduction. Thus, so as to maintain and prolong the Fasciolagigantica and Amphistomes in cattle onlifespan of the efficacy of triclabendazole currently in use traditional, small-scale dairy and large scale dairythe following recommendations are forwarded. Strict farms in the Southern Highlands of Tanzania. Tropsupervision on the usage of Triclabendazole drugs should Animal Health Prod, 37: 303-314.be implemented. Professionals and livestock owners 6. Rojo-Vázquez, F.A., A. Meana, F. Valcárcel andshould be well aware of about Triclabendazole drugs and M. Martínez-Valladares, 2002. Update on trematodeits effectiveness. Regular monitoring for anthelmintic infections in sheep. Veterinary Parasitol., 30: 15-38.resistance in livestock production communities is 7. Boray, F.E., 2002. Personal Interview at Kuleli Militaryessential to keep a track of their efficacy. Research should High School’s Tea-Garden. Çengelköy Istanbul,be done with aim of Triclabendazole efficacy and its February, 23.resistance in Ethiopia. 8. Kumsa, B. and G. Abebe, 2009. Multiple anthelmintic

ACKNOWLEDGEMENTS Ethiopia). Tropical Animal Health and Production,

First of all I would like to express my deepest thanks 9. Greenberg, R.M., 2005. Ca Signaling, voltage-gatedto God for his permission to do my daily activities as well. Ca channels and Praziquantel in flatwormNext, I would like to express my sincere and deepest neuromusculature. Parasitology, 131: 97-108.gratitude to my advisors Dr. Motuma for scholarly 10. Joseph, C.B., 2007. Liver fluke diseases in sheep andguidance, intellectual advice, constructive comments, cattle. Primefact, pp: 446.rectifying manuscript and with continuous follow up 11. Alvarez, L., G. Moreno, L. Moreno, L. Ceballos,throughout the study period in writing this paper. In third L. Shaw, I. Fairweather and C. Lanusse, 2009.place, I have no enough words to express my great full Comparative assessment of albendazole andpretty thanks, sincere appreciation and great respect for triclabendazoleovicidal activity on Fasciola hepaticaBonga sheep farm workers for their valuable piece of eggs. Vet. Parasitol., 5: 234-243.advice, encouragement and support during sample 12. Bersisa, K. and G. Abebe, 2009. Multiplecollection. Finally and most importantly, I would like to anthelmintic resistance on a goat farm in Hawassaexpress my deepest appreciation to my family, for their (Southern Ethiopia). Tropical Animal Health andendless help, love and moral support in every situation Production, 41(4): 655-662.facing me. Special thanks go to all of my friends, for their 13. Bersissa, K., D. Etana and M. Bekele, 2010.valuable comments and encouraging ideas. Comparative efficacy of albendazole, tetramisole and

REFERENCES naturally infected goats in Ziway, Oromia Regional

1. Central Statistics Authority (CSA), 2004. The Veterinary Advances, 9(23): 2905-2911.2001/2002 Ethiopian Agricultural sample enumeration 14. Urqhuart, G.M., J. Armour, J.J. Duncan, A.M. Dunn(EASE) Executive Summary, Addis Ababa, Ethiopia, and F.W. Jennings, 2007. Veterinary Parasitology1-35. (2 ed.). Wiley, Hoboken, New Jersey, USA, pp: 307.

2. Abebe, W. and G. Esayas, 2001. Survey of ovine and 15. Tagesu, A., 2017. Review on Ovine Fasciolosis incaprine gastro-intestinal helminthosis in eastern part Ethiopia. J. Vet. Sci. Res., 2(2): 000132.of Ethiopia during the dry season of the year. Revue 16. Jane, M., 2016. Current threats of triclabendazolede Medecine Veterinaire, 152(5): 379-384. resistance in F. hepatica Trends in Parasitology,

3. Biffa, D., Y. Jobre and H. Chakka, 2006. Ovine 36: 458-469.helminthosis, a major health constraint to 17. Mas-Coma, M.D. and M.A. Bargues, Valero, 2014.productivity of sheep in Ethiopia. Animal Health “Diagnosis of human fascioliasis by stool and bloodResearch Reviews, 7(1-2): 107-118. techniques: update for the present global scenario, ”

4. Sissay, M.M., A. Asefa, A. Uggla and P.J. Waller, Parasitology, 141(1): 1918-1946.2006. Assessment of anthelmintic resistance in 18. Cabada, M.M., 2016. Treatment failure after multiplenematode parasites of sheep and goats owned by courses of triclabendazole among patients withsmallholder farmers in eastern Ethiopia. Tropical fascioliasis in Cusco, Peru: a case series. PLoSNegl.Animal Health and Production, 38(3): 215-222. Trop. Dis. e10.

resistance on a goat farm in Hawassa (southern

41(4): 655-662.2+

2+

ivermectin against gastrointestinal nematodes in

State (southern Ethiopia). Journal of Animal and

nd

Am-Euras. J. Sci. Res., 15 (4): 128-139, 2020

138

19. Tadesse, D., L. Eshetu, B. Hadush, K. Amsalu and 32. HenessyMas-Coma, M.A., M.D. Valero andA. Teklu, 2014. Study on the efficacy of selectedantitrematodal drugs in naturally infected sheep withfasciolosis. Acta Parasitol Glob, 5(3): 210-213.

20. Charlier, J., 2014. Recent advances in the diagnosis,impact on production and prediction of Fasciolahepatica in cattle. Para-sitology, 141: 326-335.

21. Grocer, K.J. and J. Utzinger, 2004. Chemotherapy formajor food-borne trematodes: a review. ExpertOpinion on Pharmacotherapy, 5(8): 1711-1726.

22. Kochapakdee, S., V.S. Pandey, W. Pralomkarn,S. Choldumrongkul, W. Ngampongsai and A.Lawpetchara, 1995. Anthelmintic resistance in goatsin southern Thailand: journal of the British VeterinaryAssociation. The Veterinary Record, 137(5): 124-125.

23. Mitreva, M., D.S. Zarlenga, J.P. McCarter andD.P. Jasmer, 2007. Parasitic nematodes - FromGenomes to control. Veterinary Parasitology,148(1): 31-42.

24. McKellar, Q.A. and F. Jackson, 2004. Veterinaryanthelmintics: old and new. Trends in Parasitology,20(10): 456-461.

25. Von Samson-Himmelstjerna, G., G.C. Coles,F. Jackson, C. Bauer, F. Borgsteede, V.Y. Cirak,J. Demeler, A. Donnan, P. Dorny, C. Epe andA. Harder, 2009. Standardization of the egg hatchtest for the detection of benzimidazole resistancein parasitic nematodes. Parasitology Research,105(3): 825.

26. Brennan, G.P., I. Fairweather, A. Trudgett, E. Hoey,M. McConville, M. Meaney, M. Robinson,N. McFerran, L. Ryan, C. Lanusse and L. Mottier2007. Understanding triclabendazole resistance.Experimental and Molecular Pathol., 82(2): 104-109.

27. Thomas, O., 2000. Triclabendazole resistant Fasciolahepatica in south-west Wales. Vet. Rec, 8: 146-150.

28. National Library of Medicine (NLM), 2018.TriclabendazoleSulfoxide.

29. Fairweather, I., 2005. Triclabendazole: new skillsto unravel an old(ish) enigma. J. Helminthol.,79: 227-234.

30. WHO, 2007. Report of the WHO Informal Meeting onuse of triclabendazole in fascioliasis control, held atWHO Headquarters, Geneva, Switzerland, October2006.

31. Apt, D., 1995. Treatment of human chronicfascioliasis with triclabendazole: drug efficacyand serologic response. Am. J. Trop. Med. Hyg.,52: 532-535.

I.N. Bargues, 2014. “Digenetic Trematodes, Chapter4: Fascioliasis, ” Advances in Experimental Medicineand Biology, 766(1): 77-114.

33. Barrera, B., J.A. Otero, E. Egido, J.G. Prieto, A. Seelig,A.I. Álvarez and G. Merino, 2012. The anthelmintictriclabendazole and its metabolites inhibit themembrane transporter ABCG2/BCRP. AntimicrobialAgents and Chemotherapy, 56(7): 3535-3543.

34. Mottier, L., L. Alvarez, I. Fairweather, C. Lanusse,2006. Resistance-induced changes in triclabendazoletransport in Fasciola hepatica: ivermectin reversaleffect. J. Parasitol., 92: 55-60.

35. Virkele, L., Q.N. Cheng, Y. Zhou and X.N. Xu, 2013.“Research progress on fascioliasis,” Chinese Journalof Parasitology & Parasitic Diseases, 31(3): 229-34.

36. MestorineSmout, M.J., A.C. Kotze, J.S. McCarthyand A. Loukas, 2010. A novel high throughput assayfor anthelmintic drug screening and resistancediagnosis by real-time monitoring of parasite motility.PLoS Neglected Tropical Diseases, 4(11): 885.

37. Shoop, T.A., 2014. Trypanosomacruzi chemicalproteomics using immobilized benznidazole.Experimental. Parasitology, 140: 33-38.

38. Wolstenholme, A.J., I. Fairweather, R. Prichard,G. vonSamson-Himmelstjerna and N.C. Sangster,2004. Drug resistance in veterinary helminths.Trends in Parasitology, 20: 469-476.

39. Toner, E., G.P. Brennan, R.E.P. Hanna, H.W.J. Edgar,L. Fairweather, 2011. Distribution of egg formation byF. hepatica following treatment with triclabendazolein the sheep host. Veterinary Parasitology, 177: 79-89.

40. Robinson, M.W., J. Lawson, A. Trudgett, E.M. Hoeyand I. Fairweather, 2004. The comparative metabolismof triclabendazole sulphoxide by triclabendazole-susceptible and triclabendazole-resistant Fasciolahepatica Parasitology Research, 92: 205-210.

41. Halferty, L., J.F. Oneil, G.P. Brennan, J. Keiser,I. Fairweather, 2009. Electron microscopical study toassess the in vitro effects of the synthetic trioxolaneOZ78 against the liver fluke, Fasciola hepatica.University of Basel Library, 1325-1337.https:/www.cambridge.org/core/terms.https://doi.org/10.1017/S0031182009990643.

42. Conville Kerboeuf, D., W. Blackhall, R. Kaminskyand G. Von Samson-Himmelstjerna, 2003.Polyglycoprotein in helminths: function andperspectives for anthelmintic treatment and reversalof resistance. International Journal of AntimicrobialAgents, 22: 332-346.

Am-Euras. J. Sci. Res., 15 (4): 128-139, 2020

139

43. Welsey, S. and L. Mooney, 2009. The comparative 54. Robles-Pérez, D., J.M. Martínez-Pérez, F.M, Rojo-efficacy of four anthel-mintics against a natural Vázquez and M. Martínez-Valladares, 2013. Theacquired Fasciola hepatica infection in hill diagnosis of fasciolosis in faeces of sheep by meanssheep flock in the west of Ireland. Vet. Parasitol., of a PCR and its application in the detection of164: 201-205. anthelmintic resistance in sheep flocks naturally

44. Lee, Y.J., 2013. The small molecule triclabendazole infected. Veterinary Parasitology, 197: 277-82.decreases the intracellular level of cyclic AMP and 55. Brockwell, Y.M., 2014. Confirmation of Fasciolaincreases resistance to stress in Saccharomyces hepatica resistant to triclabendazole in naturallycerevisiae. PLoS ONE 8. infected Australian beef and dairy cattle. Int. J.

45. Gosling, S.H., S. Scarcella, G. Virkel, C. Ceriani, Parasitol. Drugs Drug. Res., 4: 48-54.J. Rodríguez and C. Lanusse, 2009. Albendazole 56. Hanna, R.E.B., H.W.J. Edgar, S. McConnell,enantiomeric metabolism and binding to cytosolic E. Tonner, M. McConville, G.P. Brennan, C. Devine,proteins in the liver fluke Fasciola hepatica. A. Flanagan, L. Halferty, M. Meaney, L. Shaw,Veterinary Research Communications, 33(2): 163-173. D. Mafftt, M. McCoy and I. Fairweather, 2010.

46. Hodgkinson, J., 2013. Identification of putative Fasciola hepatica histological changes in themarkers of triclabendazole resistance by a genome- reproductive structures of triclabendazolewide analysis of genetically recombinant Fasciola susceptible and TCBZ-R flukes after treatment inhepatica. Parasitology, 140: 1523-1533. vivo with TCBZ and the related benzimidazole

47. Moll, L., 2000. Resistance of Fasciola hepatica derivative compound alpha. Vet. Parastol.,against triclabendazole in cattle and sheep in The 168: 240-254.Netherlands. Vet. Para-sitol., 91: 153-158. 57. Toner Kelly, W.R., 1974. McMaster egg counting

48. Abbott, K.A., M. Taylor and L.A. Stubbings, 2004. technique. Vet. Clinical Diagnosis, 2 . The BailliereSustainable worm control strategies for sheep. A Tindall Company, London. UK.technical manual for veterinary surgeons and 58. Fairweather, I., 2011. Reducing the future threat fromadvisers. Sustainable Control of Parasites in Sheep (liver) fluke: realistic prospect or quixotic fantasy?(SCOPS) eds. Vet. Parasitol., 180: 133-143.

49. Jackson, F.A.M., 2011. Comparison of two assays, a 59. Martínez-Valladares, M., 2014. Efficacy of anfaecal egg count reduction test (FECRT) and a anthelmintic combination in sheep infected withcoproantigen reduction test (CRT), for the diagnosis Fasciola hepatica resistant to albendazole andof resistance to triclabendazole in Fasciola hepatica clorsulon. Exp. Parasitol., 136: 59-62.in sheep. Vet. Parasitol., 176: 170-176. 60. Crilly, J.P., 2015. Triclabendazole-resistant liver fluke:

50. Ouellette, M., 2001. Biochemical and molecular issues and strategies. Livestock, 20: 6-14.mechanisms of drug resistance in parasites. Trop. 61. Toet, H., 2014. Liver fluke vaccines in ruminants:Medical Institution Health, 6: 874-882. strategies, progress and future opportunities.

51. Taylor, M.A., K.R. Hunt and K.L. Goodyear, 2002. Institution Journal Parasitology, 44: 915-927.Anthelmintic resistance detection methods. 62. Sargison, N., 2012. Diagnosis of triclabendazoleVeterinary Parasitology, 103(3): 183-194. resistance in Fasciola hepatica. Vet. Rec.,

52. Fox, N.J., 2011. Predicting impacts of climate change 171: 151-152.on Fasciola hepatica risk. PLoS ONE 6:16126. 63. Caminade, C., 2015. Modelling recent and future

53. Robel-Perez, B., 2015. Screening anthelmintic climatic suitability for fasciolosis in Europe. Geospat.resistance to triclabendazole in F. hepatica isolated Health, 9: 301-308.from sheep by means of an egg hatch assay. BMC.Vet. Research.

nd